Gangliosides are a class of molecules which are glycolipids. Different gangliosides have been identified as prominent cell surface constituents of various transformed cells, including melanoma, as well as other tumors of neuroectodermal origin. See, e.g., Ritter and Livingston, et al., Sem. Canc. Biol., 2:401-409 (1991) and Oettgen, VCH Verlags Gesellschaft (Weinheim Germany 1989), both of which are incorporated herein by reference.
Gangliosides are known as mono-, di-, tri or polysialogangliosides, depending upon the degree of glycosylation with sialic acid residues. Abbreviations employed to identify these molecules include "GM1", "GD3", "GT1", etc., with the "G" standing for ganglioside, "M", "D" or "T", etc. referring to the number of sialic acid residues, and the number or number plus letter (e.g., "GT1a"), referring to the binding pattern observed for the molecule. See Lehninger, Biochemistry, pg. 294-296 (Worth Publishers, 1981); Wiegandt, Glycolipids: New Comprehensive Biochemistry (Neuberger et al., ed., Elsevier, 1985), pp. 199-260.
The monosialoganglioside GM3 has the structure:
2.alpha.NeuAc.fwdarw.3Gal.beta.1.fwdarw.4GK.beta.1-ceramide
The gangliosides are prevalent cell surface markers on transformed cells, such as melanoma. This has made them attractive targets for cancer research. Livingston, et al., Proc. Natl. Acad. Sci. USA, 84:2911-2915 (1987), which is incorporated herein by reference, describe results of a vaccine based trial, wherein subjects afflicted with melanoma received, as vaccines, either whole cells which present high levels of GM2, pure GM2 or pure GM2 plus bacterial adjuvant. Attention is drawn to Livingston, et al., J. Clin. Oncol., 12(5):1036-1044 (1994), and Irie, et al., U.S. Pat. No. 4,557,931, both of which are incorporated herein by reference, and deal with the use of GM2 as a vaccine.
There are difficulties unique to the immunology of gangliosides, which are touched upon briefly here. First, while these molecules are prevalent on transformed cells, they are also common on certain normal cells, such as neural cells. There is a risk, in administering gangliosides to a subject, that the resulting antibody response will damage normal cells. Indeed, certain autoimmune pathologies, such as Guillain-Barre' Syndrome, are characterized by autoimmune antibodies reactive with GM1 or GQ1b. See, e.g., Yuki, et al., J. Exp. Med., 178:11771-1775 (1993); Aspinall, et al., Infect & Immun., 62(5):2122-2125 (1994).
There is an additional practical problem in that highly pure gangliosides are extremely difficult to secure in amounts sufficient for immunization protocols. No practical synthetic method is presently available. As a result, gangliosides are secured via purification from tissue, such as bovine cranial tissues. Even under optimum conditions, the yields of pure gangliosides, including GM2 and GM3, are vanishingly small. Further, purification from mammalian tissue carries with it the risk of transmitting contaminants such as viruses, prion particles, and so forth. Alternate methodologies for securing ganglioside specific antibodies are thus highly desirable.
Due to the importance of gangliosides, it is desirable to develop a method of synthesizing high yields of pure gangliosides. The inventors of the instant application have developed novel methods of synthesizing pure GM2s, in high yields. Other methods of developing synthetic GM2s are described in Hasegawa et al., J. Carbohydrate Chemistry, 11(6):699-714 (1992) and Sugimoto et al., Carbohydrate Research, 156:C1-C5 (1986). The invention described herein develops the art in that the methods described herein are not suggested by these references.
Synthesis of GM2 can be expedited if GM3 is used as a starting material; however, GM3 is difficult to obtain from the source materials described supra, for all of the reasons given. Hence, it is desirable to have a process available which facilitates large scale production of GM3, which is free of the problems set forth supra. The invention relates, inter alia, to a process which meets these goals.